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International Journal of Research Studies in Biosciences (IJRSB)
Volume 3, Issue 3, March 2015, PP 1-15
ISSN 2349-0357 (Print) & ISSN 2349-0365 (Online)
www.arcjournals.org
©ARC Page | 1
Cloning and Characterisation of Orthologous Genes Encode
Growth Hormone in Egyptian Holstein Crossbred and Ossimi
Sheep
Waleid Mohamed El-Sayed Shakweer*
Animal Production Dept. National Research Centre
Dokki, Giza, Egypt [email protected]
Yassein Mohamed Hafez
Animal Production Dept. Faculty of Agriculture,
Cairo University, Giza, Egypt
Marwa Naguib Mohamed Esmaail Sanad
Genetic and Cytology Dept.
National Research Centre Dokki, Giza, Egypt
Ashraf Abd El-Halim El-Sayed
Animal Production Dept.
Faculty of Agriculture Cairo University, Giza, Egypt
Ibrahim Mohamed Awadalla
Animal Production Dept.
National Research Centre
Dokki, Giza, Egypt [email protected]
Mamdouh Ibrahim Mohamed
Animal Production Dept.
National Research Centre
Dokki, Giza, Egypt [email protected]
Abstract: This study aims to investigate the compatibility between growth hormone (GH) cDNA sequence
in Egyptian cows and sheep as an introduction to produce bovine GH-Transgenic sheep using sperm
mediated gene transfer (SMGT) technique to improve their productivity. Our previous in vivo study indicates a significant increase in the concentration of GH mediator (IGF-1) in blood, and daily growth
rate, when Egyptian rams were injected with bovine somatotropin. The GH gene of Egyptian Holstein
crossbred (EH_GH) and Ossimi sheep breed (Os_GH) has been characterized in this study. The mRNA of
pituitary gland has been isolated and then the cDNA was cloned into pTZ57R/T cloning vector. The
complete coding domain sequences (CDSs) of EH_GH (AC: KP221576) AND Os_GH (Ac: KP221575)
were detected and annotated comprehensively. The sequenced GH genes varied slightly at gene length;
EH_GH was 740 bp instead of 690 bp in Os_GH, but with similar gene structure (5 exons and 4 introns).
High homology (98%) in GH protein length and structure was observed; the predicted open reading frames
(ORF) length was 217 aa and 216 aa of EG_GH and Os_GH, respectively, which included only four
different amino acids in the predicted ORFs. A gene synteny was developed of the homologous bovine and
ovine GH1 genes Ensemble annotated genome database. The results showed strong conserved structure and function, that indicated to GH gene of Egyptian Holstein crossbred and Ossimi breed are orthologous
genes. We assume in this study that the novel genetic information about the bovine and ovine GH in
Egyptian breeds can support; further success possibilities of producing the bovine GH-transgenic sheep to
improve the growth and milk production performance.
Keywords: Growth hormone (GH), Orthologous genes, Egyptian Holstein crossbred, Ossimi breed.
1. INTRODUCTION
Sheep is one of the most important domestic animals reared in Egypt. There are almost 5.5
million head that contributed about 6% of the total red meet that produced in Egypt [1]. The cows'
population in Egypt is estimated about 4.53 million heads that produced 2.90 and 0.41 million tons of milk and meat, respectively [2]. In the last three decades, Egyptian farms adopted crosses
between the Holstein breed and the Egyptian Baladi breeds to improve their productivity by
producing Egyptian Holstein crossbred adapted in the Egyptian environmental. Due to a shortage of animal production in the world, a lot of scientists have been turned on to gene transfer
techniques as a quick tool to increase livestock production. This study aims to investigate the
Waleid Mohamed El-Sayed Shakweer*
International Journal of Research Studies in Biosciences (IJRSB) Page | 2
compatibility between the growth hormone cDNA sequence in Egyptian cows and sheep as an
introduction to produce bovine GH-Transgenic sheep using sperm mediated gene transfer (SMGT) to improve their productivity. The candidate gene is growth hormone that spans
approximately 1.8 kilobases and consists of five exons interrupted by four introns [3]. Growth
Hormone gene is located at chromosome 19 in Bos taurus genome, while at chromosome 11 in
Ovis aries genome [4] and [5]. The growth hormone is a single-chain polypeptide synthesised and secreted by the pituitary gland under hypothalamic control and regulates the body growth, cell
reproduction and regeneration in cattle and sheep [6]. Growth hormone affects the partitioning of
nutrients among tissues in sheep and cattle, increasing bone growth and milk production and decreasing fatness [7]. It belongs to the same hormonal family as prolactin (PRL) and placental
lactogen (PL), all of them sharing certain biological, immunological and structural features; they
are considered to be derived from duplication of a single ancestral gene [8] and [9]. Plasma
concentration of GH is increased in cows genetically selected for high milk production [10] and in sheep selected for low levels of fatness [11], indicating that GH is genetically correlated with
these traits.
In our previous study, recombinant bovine somatotropin (100 mg rbST/14 days) has been injected in Egyptian Rahmani lambs. The average daily gain has been increased significantly (8.43%,
P<0.05) in rbST treated rams comparing with the control. The feed conversion did not differ
significantly in rbST treated rams compared to the control that may be gives us an indicator that the treated group utilizes the feed intake in a good way to increase the average daily gain [12].
Based on that aforementioned, our aim was cloning and characterization of growth hormone gene
in both Egyptian Holstein crossbred and Ossimi sheep breed. The functional bioinformatic
analysis can guide the research accurately to predict for the success possibilities to introduce and express bovine GH gene into transgenic sheep for enhancing their productivity.
2. MATERIALS AND METHODS
2.1. cDNA Synthesis
Total RNA has been isolated from pituitary gland (50 mg) of Egyptian Holstein crossbred and
Ossimi breed using Booze reagent kit (Bioflux®). Total RNA was reverse transcribed with Oligo
dT18 using RevertAid First Strand cDNA Synthesis Kit (Fermentas, Canada). The EH_GH primer was designed using custom primers-OligoPerfect™ designer tool (https://tools. lifetechnologies.
com/content.cfm?pageid=9716&icid=fr-oligo-6?CID=fl-oligoperfect) based on NCBI database
(Ac: M27325.1, 783bp). The EH_GH primer sequence was EH_GH-F 5'-GAGCTCCAGGGTCCTGTGGACAGC-3' and EH_GH-R 5'-CCGCGGTGCGATGCAATTTCC
TCAT-3'. While, the Os_GH primer was published on NCBI (Ac: EU935861.1), Os_GH-F 5'-
GCTCACCAGCTATGATGGCTG-3' and Os_GH-R 5'-TGGCAACTAGAAGGCGCAGCT-3'.
2.2. Gene Cloning
The PCR reaction mixture (10µl) was prepared for each gene as follow; [1µl of 10X buffer
containing MgCl2 (25mM), 1µl of dNTBase mixture (200 µM), 1µl of F-primer, 1µl of R-primer,
0.2 µl of Taq polymerase (1U/µl), 1µl of both EH_GH/Os_GH cDNA and water nuclease-free up to 10µl) [13]. The amplification reaction was carried out in a Thermocycler PCT-100 (MJ
Research, USA). The PCR conditions of EH_GH-cDNA was denaturation for 1 min at 94ºC,
annealing for 2 min at 57ºC, and extension for 3 min at 72ºC for 29 cycles, and final extension at 72ºC for 7 min, while for Os_GH-cDNA, denaturation for 1 min at 94ºC, annealing for 2 min at
63ºC, and extension for 3 min at 72ºC for 29 cycles. A final extension was 72ºC for 7 min. The
PCR products were visualized on a 1% agarose gel stained with Ethidium bromide in 1x TAE
buffer at 100 v for 60 min with 10 Kb BioLabs ladder, (New England, UK). The image of the gel band was taken using Bio-Rad Gel Doc XR. The detected EH_GH and Os_GH amplicons were
extracted from the gel using GeneElute™ Gel Extraction Kit (Fermentas, USA), then cloned into
pTZ57R/T vector (Fig.1). The vector construction was designed according to the cloning kit procedure (Fermentas, USA) as follow; 3µl of pTZ57R/T vector, 2.5µl of both EH_GH/Os_GH
cDNA, 6µl of 10x ligase buffer, 1µl T4 DNA ligase and 17.5µl sterile water in total 30 µl reaction
mixture, incubated at 22°C/1h for ligation. The ligated product was transformed into DH5α cells (Fermentas, USA). The constructed plasmids (EH_GH and Os_GH-PTZ57R/T) were extracted
using GeneJET plasmid Miniprep kit, and sequenced. The efficiency of transformation was
determined using the polymerase chain reaction.
Cloning and Characterisation of Orthologous Genes Encode Growth Hormone in Egyptian Holstein
Crossbred and Ossimi Sheep
International Journal of Research Studies in Biosciences (IJRSB) Page | 3
Fig1. Map of the pTZ57R/T cloning vector.
2.3. Bioinformatics Analysis
The consensus sequence of cloned EH_GH and Os_GH cDNA were created using CodonCode
Aligner V4.2.7 software. The SNPs between EH_GH and Os_GH cDNA sequence have been
done using the pairwise alignment via EMBOSS Needle tool (http://www.ebi.ac.uk/Tools/psa/
emboss_needle/). The transcription promoter has been predicted using Neural Network Promoter Prediction via http://www.fruitfly.org/seq_tools/promoter.html [14]. The open reading frames
(ORF) of EH_GH and Os_GH cDNA sequences were predicted using http://web.expasy
.org/translate/ [15]. Predicted protein codons relative to the nucleotide sequence were done using translated tool via http://www.fr33.net/translator.php. The predicted cleavage site in signal
peptide sequence was done using SignalP 4.1 Server tool http://www.cbs.dtu.dk /services/SignalP/
[16]. The conserved domain of EH_GH and Os_GH cDNA sequences were detected using NCBI conserved domains tool via http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi [17]. The motif
of EH_GH and Os_GH cDNA was predicted using search motif library tool at http://www.
genome.jp/tools/motif/. The cysteine state and disulfide bonds in the EH_GH and Os_GH protein
sequences were carried out using cysteine and disulfide bond prediction tool at http://clavius.bc. edu/~clotelab/DiANNA/ [18]. Pairwise alignment was done between EH_GH and Os_GH protein
sequences using EMBOSS Needle tool (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Also,
the multiple alignments of EH_GH and Os_GH protein sequences vs. NCBI protein database have been carried out using Bioedit 7.2.5 software [19]. Phylogenetic of EH_GH and Os_ GH protein
sequences was conducted using MEGA version 6 [20]. The synteny location of GH in ovine
chromosome (Chr11) vs. bovine chromosome (Chr19) has been achieved using annotation and
prediction tool available at http://www.ensembl.org.
3. RESULTS AND DISCUSSION
3.1. Gene Cloning of GH
The cDNA and translated protein sequences of EH_GH and Os_GH have been published at
Genebank-NCBI database with accession number KP221576 and KP221575, respectively. The
electrophoresed band of EH_GH was 740 bp and was 690 bp for Os_GH (Fig. 2). In Fig. 3, five
uncut plasmid forms were showed on one sample of EH_GH-TZ57R/T, whereas the Os_GH-TZ57R/T had three to four forms. About five forms of uncut plasmid on the electrophoretic gel
based on its form and molecular weight; 1) Nicked open-circular 2) Relaxed circular 3) Linear, 4)
Supercoiled, 5) Supercoiled denature [21]. The electrophoresis results of EH_GH-TZ57R/T and Os_GH-TZ57R/T are shown in Fig. 4.
Waleid Mohamed El-Sayed Shakweer*
International Journal of Research Studies in Biosciences (IJRSB) Page | 4
Fig2. Gel electrophoresis of cDNA band of EH_GH and O_GH.
Fig3. Photograph of Holstein and Ossimi GH-TZ57R/T uncut plasmid electrophoresis.
Fig4. Photograph of EH_GH-TZ57R/T and Os_GH-TZ57R/T PCR plasmid electrophoresis.
Fig5. Photograph of EH and Os_GH-TZ57R/T cut plasmid.
Cloning and Characterisation of Orthologous Genes Encode Growth Hormone in Egyptian Holstein
Crossbred and Ossimi Sheep
International Journal of Research Studies in Biosciences (IJRSB) Page | 5
3.2. Growth Hormone cDNA SNPs
The pairwise alignment (Fig. 8) has been shown that there are ten nonsynonymous alleles
substitutions (small letter) which yield a new codon that encodes a different amino acids in EH_GH cDNA sequence vs. Os_GH cDNA sequence (Table 1); gct for Alanine (Ala) at 34,35
and 36, ccc for Proline (Pro) at 43, 44 and 45, Cgc for Arginine (Arg) at 650 and 651, gcc for
Alanine (Ala) at 688, 689 and 690, atC for Isoleucine (Ile) at 691 and 692, tGt for cysteine (Cys) at 694 and 696, tgT for Cysteine (Cys) at 697 and 698, Ttg for Leucine (Leu) at 701 and 702, ccc
for Proline (Pro) at 703, 704 and 705 and ccc for Proline (Pro) at 709, 710 and 711in EH_GH
cDNA sequence vs. aag for Lysine (Lys) at 12, 13 and 14, aCC for Threonine (Thr) at 21, Cag for Glutamine (Glu) at 628 and 629, aaa for Lysine (Lys) at 666, 667 and 668, cgC for Arginine
(Arg) at 669 and 670, cGg for Arginine (Arg) at 772 and 774, ctT for leucine (Leu) at 675 and
676, Tgc for Cysteine (Cys) at 780, tgg for Tryptophan (Trp) at 681, 682 and 683 and ggg for
Glycine (Gly) at 687, 688 and 689 in Os_GH cDNA sequence.
Fig6. The growth hormone cDNA sequence and the encoded amino acid residues in Egyptian Holstein
crossbred. 1Signal protein sequence (Red, 1…27 aa), 2Conserved domain of predicted residues sequence
(Yellow: 36…215 aa), 3First motif Somatotropin_1 (Green: 79…112, 192 related sequences and 22 related
structures). 4Second motif Somatotropin_2 (Green: 190...207, 194 related sequences and 22 related
structures). 5Predicted promoter (Purple, start 603 bp....end 653 bp and score 0.85). The conserved
Cysteine residues are illustrated by boxes.
Waleid Mohamed El-Sayed Shakweer*
International Journal of Research Studies in Biosciences (IJRSB) Page | 6
Fig7. The growth hormone cDNA sequence, predicted amino acids sequences and protein features in
Ossimi breed. 1
Signal protein sequence (Red, 1…26 aa), 2
Conserved domin of predicted residues sequence
(Yellow: 35…214 aa), 3
First motif Somatotropin_1 (Green: 78...111, 192 related sequences and 22 related
structures). 4
Second motif Somatotropin_2 (Green: 189…206, 194 related sequences and 22 related
structures). 5
Predicted promoter (Purple, start 581bp... end 631bp and score 0.85). The conserved Cysteine
residues are illustrated by boxes.
Fig8. The pair wise alignment of EH and Os_GH cDNA sequences.
On the other hand, there are five synonymous allele’s substitutions (small letter) which yield a
new codon that encodes the same amino acids TGt for Cysteine (Cys) at 672, TTc for
Phenylalanine (Phe) at 678, TAg Stop codon at 681, CCa for Proline (Pro) at 687 and cTc Leucine (Leu) at 706 and 708, in EH_GH cDNA sequence vs. TGc for Cysteine (Cys) at 650, TTt
for Phenylalanine (Phe) at 656, TAa Stop codon at 659, CCc for Proline (Pro) at 665 and tTg
Leucine (Leu) at 684 and 686 in Os_GH cDNA sequence. That single nucleotide polymorphism can be used to develop SNPs markers for each breed.
3.3. The Prediction of Growth Hormone Promoter
The promoter is the most important regulatory region that regulates the very first step of gene
expression [22]. The promoter sequence of EH_GH cDNA was AAGGACCTGCATAAGACGG AGACGTACCTGAGGGTCATGAAGTGCCGCC (50bp), contains start codon (ATG) and
spanning between 603bp and 653bp with prediction score 0.85 (Fig. 6). Whereas the promoter of
Os_GH was GAAGGCATCCTGGCCCTGATGCGGGAGCTGGAAGATGG (38bp), contains start codon (ATG) and spanned between 581bp to 631bp with 0.85 prediction score (Fig. 7). The
identity score between both promoters was 57.89 considering length differences.
3.4. Protein Structure
3.4.1. Signal Peptide Sequence
The signal peptide of EH_GH sequence ranged from 1 to 27 residues and from 1 to 26 residues in
Os_GH cDNA (Fig. 6 and 7, respectively). The predicted cleavage site was located between 26
and 27 amino acids in EH_GH protein and between 25 and 26 amino acids in Os_GH protein (Fig. 9). The signal peptide has been defined as a sequence generally found at the N-terminal end
of most secreted and cell-surface protein precursors [23]. The signal sequence that bound at the
amino terminus of the emerging protein “flag”, which transport the mechanism of the cell to prompt them as to where the emerging protein should go. The flags recognize by signal
recognition particle (SRP) locate freely in the cytoplasm or attached to the ribosome, the SPR
Cloning and Characterisation of Orthologous Genes Encode Growth Hormone in Egyptian Holstein
Crossbred and Ossimi Sheep
International Journal of Research Studies in Biosciences (IJRSB) Page | 7
interacts with the signal sequence and leads to the transient arrest of translation. That may indicate
that the isolated GH cDNA of both breed could be expressed in transfected cell [24].
Table1. The modified nucleotides (SNPs) and encoded amino acids in EH_GH cDNA vs. Os_GH cDNA sequences.
EH_GH cDNA sequence Os_GH cDNA sequence
Nucleotides Location Residue Nucleotides Location Residue *gct 34, 35 and 36 Alanine aag 12, 13 and 14 Lysine
ccc 43, 44 and 45 Proline aCC 21 Threonine
Cgc 650 and 651 Arginine Cag 628 and 629 Glutamine
TGt 672 **Cysteine TGc 650 **Cysteine
TTc 678 **Phenylalanine TTt 656 **Phenylalanine
TAg 681 **Stop TAa 659 **Stop
CCa 687 **Proline CCc 665 **Proline
gcc 688, 689 and 690 Alanine aaa 666, 667 and 668 Lysine
atC 691 and 692 Isoleucine cgC 669 and 670 Arginine
tGt 694 and 696 Cysteine cGg 672 and 674 Arginine
tgT 697 and 698 Cysteine ctT 675 and 676 Leucine
Ttg 701 and 702 Leucine tgc 679 and 680 Cysteine
ccc 703, 7024 and 705 Proline tgg 681, 682 and 683 Tryptophan
cTc 706 and 708 **Leucine tTg 684 and 686 **Leucine
ccc 709, 710 and 711 Proline ggg 687, 688 and 689 Glycine
C……..C 1 to 22 Q……. P No complement nucleotide
G………A 713 to 740 R…… C No complement nucleotide
* Lower case letters have been indicated for SNPs, ** The same amino acids with different encoding
sequence
3.4.2. Conserved Domain and Motifs
Fig9. The predicted cleavage site of signal peptide in Egyptian Holstein crossbred (A) and Ossimi breed
(B). C-score: Signal peptide cleavage sites. S-score: Signal peptide positions within signal peptides from positions in the mature part of the proteins and from proteins without signal peptides. Y-score: Better
cleavage site prediction than the raw C-score alone. Mean S: The average S-score of the possible reticulum
the SRP detaches and this leads to the continuation of protein translation, and eventually to the signal
peptide (from position 1 to the position immediately before the maximal Y-score). D-score: Discriminate
signal peptides from non-signal peptides. For non-secretory proteins all the scores represented in the
SignalP output should ideally be very low (close to the negative target value of 0.1) [15].
Highly conserved domain of predicted growth hormone protein has been detected in EH_GH and
Os_GH (Fig. 10). Detecting the significant conserved protein domains is often required for basic cellular function, stability or reproduction. Conservation of protein structures is indicated by the
presence of functionally equivalent, though not necessarily identical, amino acid residues and
structures between analogous parts of proteins [25]. The predicted conserved domain hit
Waleid Mohamed El-Sayed Shakweer*
International Journal of Research Studies in Biosciences (IJRSB) Page | 8
somatotropin like hormone and specific family with growth hormone like superfamily; Interval 35
to 214 aa, 1.40e-104 E-value for Os_GH protein and Interval 36 to 215 aa, 5.30e-106 E-value for EH_GH protein. Protein motifs are signatures of protein families and can often be used as tools
for the prediction of protein function [26]. We detected two common motifs in both EH_GH and
Os_GH cDNA but with different spanning (Fig. 6 and 7); Somatotropin_1 (CFSETIPAPTGKNE
AQQKSDLELLRISLLLIQSW) from 79 to 112 aa for EH_GH cDNA and from 78 to 111 aa for Os_GH cDNA. Somatotropin_2 (CFRKDLHKTETYL RVM KC) from 190 to 207 aa for EH_GH
cDNA and from 189 to 206 aa for Os_GH cDNA which reflects same function and
characteristics. A comprehensive summary of EH_GH and Os_GH annotation has been covered in Table (2).
Fig10. Conserved domain of EH_GH and Os_GH protein sequences.
Table2. Comprehensive summary of EH_GH and Os_GH sequences annotation.
Parameters EH_GH cDNA Os_GH cDNA
Chromosome location 19 11
Exon number 5 5 Exon protein position 1..31..150..175..205 1..30..149..175..204
Intron number 4 4
Length of mRNA 740 bp 690 bp
Predicted promoter Start 603, End 653, Score 0.85 Start 581, End 631, Score 0.85
Motifs
Somatotropin_1 From 79 to112 From 78 to 111
Somatotropin_2 From 190 to 207 From 189 to 206
Molecular weight 24558 Da 24460 Da
Number of residues 217 216
Disulfide bonds 79 - 190 78 - 189
207 - 215 206 - 214
Cysteine position
18 17
79 78
190 189
207 206
215 214
Physical characteristics of growth hormone protein
Length of protein 217 aa 216 aa
Chain peptide 27-217 (190 aa) 26-216 (190 aa)
Open reading frame 1-217 1-216
Signal peptide 1-27 1-26
Conserved domain
n 36-215 35-214
Cloning and Characterisation of Orthologous Genes Encode Growth Hormone in Egyptian Holstein
Crossbred and Ossimi Sheep
International Journal of Research Studies in Biosciences (IJRSB) Page | 9
3.4.3. Cysteine Bridge and Disulfide Bounds
Five conserved cysteine (Cys) are founded in both EH_GH and Os_GH protein sequence at the
following positions; (Cys18
, Cys79
, Cys190
, Cys207
and Cys215
) and (Cys17
, Cys78
, Cys189
, Cys206
and Cys
204), respectively residues (Fig 6 and 7). Two sulfide bounds are predicted between 79 and 190
aa (99%) and between 207 and 215 aa (98%) in EH_GH protein and between at 78 and 189 aa
(99%) and between 206 and 214 aa (93%) in Os_GH protein. The disulfide bonds play an important role in the folding and stability of some proteins, usually proteins secreted to the
extracellular medium [27]. Four cysteine residues from five have been participated in the
formation of two disulfide bonds between the thiol groups of cysteine residues by the process of oxidative folding. The disulfide bond stabilizes the folded form of a protein in several ways; 1) It
holds two portions of the protein together, biasing the protein towards the folded topology. 2) The
disulfide bond may form the nucleus of a hydrophobic core of the folded protein [28]. One
possible role of this fifth Cysteine residue might be the formation of oligomeric complexes, which might affects the appropriate re-folding of growth hormone protein [29]. In the intracellular
environment cysteines can still play a key structural role. Their sulphydryl side chain is excellent
for binding to metals, such as zinc, meaning that cysteines are very common in metal binding motifs such as zinc fingers [30].
3.4.4. Pair wise alignment of Open Reading Farm
The pairwise alignment of EH_GH protein vs. Os_GH protein (Fig. 11) has been indicated that the protein sequences in both breeds are highly identical residues (98%) except in four positions
(Table 3). The EH_GH protein sequence was started with two start codon (MM), while the
Os_GH protein sequence was started with unusual single start codon (M). The modified residues
of EH_GH protein sequence were Ala3, Pro
6 (signal peptide) and Arg
208 vs. Lys
2, Thr
5 (signal
peptide) and Gln207
in Os_GH protein sequence. The differences in residues substitution in signal
peptide did not affect translocation efficiency and cleavage site position and this conclusion is
confirmed based on the results of highly values of C, Y, S and D in signalP chart (Fig. 9). But the difference in the N-terminus Methionine can be higher growth hormone transcription in EH_GH,
and lower expression in Os_GH, refer to the active and inactive forms of GH [31].
Fig11. The pairwise alignment between EH_GH and Os_GH protein sequences
The Ala and Lys can substitute together because both belong to the same amino acid group (small) as well as Arg vs. Gln both are polar residues. Although, the Pro is a small residue and Thr is a polar residue, in addition to a common role of Pro and Thr in facilitating the intracellular signal transduction but they can substitute without negative effect on protein function [15], [32] and [33]. It may worth to look into the unique residue in Os_GH (Lys) in exon2, which was associated with a mutation (AAG) vs. (Ala, GCT), which is a common codon among all the aligned proteins, and see if the mutation effected on the receptor efficiency especially with the missing initial Met in Ossimi. In respect to the important role of Lys in structure and locate on the outside of the protein, while Ala rarely involved in protein function directly but can play a role in substrate recognition or specificity [33]. There is about 97.5% homology in the coding regions between bovine GH and ovine GH genes [34]. The GH homology can supports and explain the assumption the expression possibility of bovine growth hormone in injected sheep breed as mentioned previously in rams [12], [35], [36], [37] and [38].
Waleid Mohamed El-Sayed Shakweer*
International Journal of Research Studies in Biosciences (IJRSB) Page | 10
3.4.5. Multiple Alignment
Gene families arise by gene duplication and natural selection [39]. The multiple alignments are an
essential study to understand the evolution between the bovine and ovine growth hormone
precursors. The multiple alignment of EH_GH and Os_GH against other GH complete CDS
(Genebank- NCBI database), and the main published GH1 of bovine and ovine (Ensemble
genomes database) were shown in Fig. 12. There are three pakistanian ovine breeds (Latti, Lohi
and Afghani) and four bovine breeds (Indian Zeubo, Iranian cattle, Pakistani Sahiwal cattle and
Table3. Substituted residues of growth hormone protein of Egyptian Holstein crossbred and Ossimi breed
vs. investigated NCBI breeds.
Studied breeds Modified residues
Investigated breeds Residue Position Residue Position
Egyptian
Ossimi breed
Glycine 34 Serine 35 Afghani breed, Pakistan
Leucine 106 Proline 107 Dhanni cattle, Pakistan
Glutamine 109 Arginine 110
Phenylalanine 122 Leucine 123 Lezhi Black breed Capra
hircus, China
Glutamic acid 152 Glycine 153 Tibetan ovine breed, China
Glycine 155 Valine 156 All tested breeds except
Afghani ovine breed
Tyrosine 167 Histamine 168 Latti ovine breed, Pakistan
Glutamine 207 Arginine 208 All tested breeds database
Egyptian
Holstein
crossbred
Leucine 107 Proline 107 Dhanni cattle, Pakistan
Glutamine 110 Arginine 110
Glycine 156 Valine, 156 Kamori cattle, Pakistan
Leucine 189 Phenylalanine 189 Irani cattle
Egyptian
Holstein
crossbred
Methionine 1 - 1
Egyptian Ossimi breed Alanine 3 Lysine 2
Proline 6 Thrionine 5
Arginine 208 Glutamine 207
Chinese cattle) have partial sequences (four exons) while in Zebu cattle contains five exons but
the first exon is very short contains only four residues. In most mammals, GH sequence is
strongly conserved, but differences in the biological and receptor-binding properties due to the
species-specificity of receptor-binding [40]. Interestingly, the results of multiple alignment have
been illustrated that there is a distinct mutation in growth hormone protein sequence where the
conserved Gly156
has been substituted with Val156
in following breeds; Ovis aries ref synteny,
Pakistani Kamori cattle, Pakistani Latti ovine, Chines Tibetan ovine, Pakistani lohi ovine, Chines
Kazakh ovine, Swiss Saanen goat, Portugal Serra da Estrela ovine, Chines Lezhi Black goat,
Chines Tibet goat and Chines ovine compared to the other breeds (Table 3). Normally, if the
conserved Glycine has been changed to any other amino acid, this change could have a drastic
impact on protein function [33]. Therefore, it could be concluded that it is possible to substitute
conserved Gly and get a functional growth hormone protein, this mutation may be considered as a
specific genetic marker related to specific breed in specific region especially in China and
Pakistan based on the investigated breeds. The Ossimi breed contains distinct modified residues;
Lys2 and Thr5 vs. Ala
3 and Pro
6 in all investigated breeds, respectively (Table 3). The substitution
of Pro6 to Thr
6 and Ala
3 to Lys
2 are located out open reading frame therefore did not affect the
protein function [32], [33] and [41]. In the present study, the Gly35
residue was presented in all
investigated breeds vs. Ser35
residue in Afghani sheep breed. Because the both Gly and Ser are
tiny and small residues and the Gly is not in the conserved domain sequence; therefore the
substitution is functional [32]. The substitution of Phe123, 217
and Leu188
in all tested breeds with
Leu123, 217
and Phe188
in Lezhi Black goat, Dhanni x Friesian cattle crossbred and Iranian cattle
breed, respectively are acceptable and may use this substitution as a distinct genetic marker in
Lezhi Black goats and in Iranian cattle breed. In the present study, the Glu154
residue is presence
in all breeds vs. Gly154
in Pakistani Afghani ovine breed. This mutation may causes reduction in
growth hormone function in this breed resulting in the differences between them in physio-
chemical properties. The Tyr168
residue is presence in all tested breeds vs. His168
residue in
Pakistani Latti ovine breed. Because the both histidine and tyrosine are polar amino acids
(neutral), therefore Tyr can substitute with His residue [32].
Cloning and Characterisation of Orthologous Genes Encode Growth Hormone in Egyptian Holstein
Crossbred and Ossimi Sheep
International Journal of Research Studies in Biosciences (IJRSB) Page | 11
10 20 30 40 50 60 70 80 90 100
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Egyptian_Holstein_croosbred MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Egyptian_Ossimi_breed_oGH_prot -MKAGTRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Bos_taurus_ref_synety_somatotr MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Ovis aries_ref_synety_somatotr MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Zeubo_bGH_Indian -------------------------MAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Chinese_cattle_bGH_|EF470979.1 ------------------------------MSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Sahiwal_breed_bGH1_Pakistan ------------------------------MSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Nelor_strain_bGH_Brasil MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Bovine_GH_|M27325.1| MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
bGH_Iran ------------------------------MSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Kamori_breed_bGH_Pakistan MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Dhanni_breed_bGH_Pakistan MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
DhannixFriesian_crossbred_bGH MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Red_Sindhi_breed_bGH_Pakistan MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Thari_breed_bGH_Pakistan MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Mongolian_breed_bGH_China MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Kenana_breed_bGH_Sudanese MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Butana_breed_bGH_Sudanese MMAAGPRTSLLLAFALLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Latti_breed_oGH_precursor_Paki ------------------------------MSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Afghani_breed_oGH_Pakistan ------------------------------MSLSSLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Tibetan_breed_oGH_China MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
lohi_breed_oGH_Pakistani ------------------------------MSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Kazakh_breed_oGH_China MMAAGPRTSLLLAFTMLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Saanen_strain_Capra hircus_GH_ MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Serra_da_Estrela_breed_oGH_Por MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Lezhi_Black_breed_Capra_hircus MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
Tibet_breed_Capra_hircus_China MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
oGH_protein_China MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLE
110 120 130 140 150 160 170 180 190 200....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
Egyptian_Holstein_croosbred LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Egyptian_Ossimi_breed_oGH_prot LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Bos_taurus_ref_synety_somatotr LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Ovis aries_ref_synety_somatotr LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDRNMRSDDALLKNYGLLSCFRKDLHKTET
Zeubo_bGH_Indian LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Chinese_cattle_bGH_|EF470979.1 LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Sahiwal_breed_bGH1_Pakistan LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Nelor_strain_bGH_Brasil LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Bovine_GH_|M27325.1| LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
bGH_Iran LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLFSCFRKDLHKTET
Kamori_breed_bGH_Pakistan LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Dhanni_breed_bGH_Pakistan LLRISLPLIRSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
DhannixFriesian_crossbred_bGH LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Red_Sindhi_breed_bGH_Pakistan LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Thari_breed_bGH_Pakistan LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Mongolian_breed_bGH_China LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Kenana_breed_bGH_Sudanese LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Butana_breed_bGH_Sudanese LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Latti_breed_oGH_precursor_Paki LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTHDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Afghani_breed_oGH_Pakistan LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Tibetan_breed_oGH_China LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELGDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
lohi_breed_oGH_Pakistani LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Kazakh_breed_oGH_China LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Saanen_strain_Capra hircus_GH_ LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Serra_da_Estrela_breed_oGH_Por LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Lezhi_Black_breed_Capra_hircus LLRISLLLIQSWLGPLQFLSRVLTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
Tibet_breed_Capra_hircus_China LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
oGH_protein_China LLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTET
210....|....|....|..
Egyptian_Holstein_croosbred YLRVMKCRRFGEASCAF
Egyptian_Ossimi_breed_oGH_prot YLRVMKCQRFGEASCAF
Bos_taurus_ref_synety_somatotr YLRVMKCRRFGEASCAF
Ovis aries_ref_synety_somatotr YLRVMKCRRFGEASCAF
Zeubo_bGH_Indian YLRVMKCRRFGEASCAF
Chinese_cattle_bGH_|EF470979.1 YLRVMKCRRFGEASCAF
Sahiwal_breed_bGH1_Pakistan YLRVMKCRRFGEASCAF
Nelor_strain_bGH_Brasil YLRVMKCRRFGEASCAF
Bovine_GH_|M27325.1| YLRVMKCRRFGEASCAF
bGH_Iran YLRVMKCRRFGEASCAF
Kamori_breed_bGH_Pakistan YLRVMKCRRFGEASCAF
Dhanni_breed_bGH_Pakistan YLRVMKCRRFGEASCAF
DhannixFriesian_crossbred_bGH YLRVMKCRRFGEASCAL
Red_Sindhi_breed_bGH_Pakistan YLRVMKCRRFGEASCAF
Thari_breed_bGH_Pakistan YLRVMKCRRFGEASCAF
Mongolian_breed_bGH_China YLRVMKCRRFGEASCAF
Kenana_breed_bGH_Sudanese YLRVMKCRRFGEASCAF
Butana_breed_bGH_Sudanese YLRVMKCRRFGEASCAF
Latti_breed_oGH_precursor_Paki YLRVMKCRRFGEASCAF
Afghani_breed_oGH_Pakistan YLRVMKCRRFGEASCAF
Tibetan_breed_oGH_China YLRVMKCRRFGEASCAF
lohi_breed_oGH_Pakistani YLRVMKCRRFGEASCAF
Kazakh_breed_oGH_China YLRVMKCRRFGEASCAF
Saanen_strain_Capra hircus_GH_ YLRVMKCRRFGEASCAF
Serra_da_Estrela_breed_oGH_Por YLRVMKCRRFGEASCAF
Lezhi_Black_breed_Capra_hircus YLRVMKCRRFGEASCAF
Tibet_breed_Capra_hircus_China YLRVMKCRRFGEASCAF
oGH_protein_China YLRVMKCRRFGEASCAF
Fig12. Multiple alignments of the encoded proteins of Egyptian Holstein crossbred and Ossimi breed vs.
tested NCBI database breeds.
3.4.6. Phylogeny Tree and Synteny
The phylogenetic tree of EH_GH and Os_GH protein sequences vs. investigated breeds has been shown in Fig. 13. The predicted synteny location between Egyptian Holstein crossbred (Chr 19)
and Ossimi breed (Chr 11) is shown in Fig. 14. Comparisons of mammalian gene maps show
large regions of conserved synteny where the same genes map to equivalent regions in different species [4], [5], [42], [43] and [44].
Waleid Mohamed El-Sayed Shakweer*
International Journal of Research Studies in Biosciences (IJRSB) Page | 12
Fig13. Phylogenetic tree of Egyptian Holstein crossbred and Ossimi breed growth hormone protein
sequences vs. tested NCBI breeds.
From phylogenetic tree and synteny results have been illustrated that EH_GH gene at
chromosome 19 is an orthologous gene to the Os_GH gene at chromosome 11, as expected from their close evolutionary relationship [45]. The results support our assumptions and our previous
work [12], and can induce a clue for further studies to produce transgenic sheep carry an
expressed copy of bovine growth hormone gene using Sperm Mediated Gene Transfer (SMGT) technique as mentioned previously in different mammalian systems [46], [47] and [48] in mice;
[49] in rabbits; [50] in pigs; [51] and [52] in Goat; [53] and [54] in sheep and [55] in cattle.
Fig14. The predicted synteny location of growth hormone protein sequences between Egyptian Holstein
crossbred (Chr 19) and Ossimi breed (Chr 11).
4. CONCLUSION
It could be concluded that GH sequence of Egyptian Holstein crossbred and Ossimi breed are orthologous genes (98%). We assume in this study that the novel genetic information about the
bovine and ovine GH in Egyptian breeds can support further success possibilities of producing
GH-transgenic sheep, using sperm mediated gene transfer (SMGT) technique to enhance their
growth performance.
ACKNOWLEDGMENTS
Many Thanks to Dr. Mamdouh Sharaf El-Deen, Professor of Poultry Production, Animal Production Department, Faculty of Agriculture, Cairo University, for his financial support of this
work.
Cloning and Characterisation of Orthologous Genes Encode Growth Hormone in Egyptian Holstein
Crossbred and Ossimi Sheep
International Journal of Research Studies in Biosciences (IJRSB) Page | 13
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